Vertical wheel bioreactors

ABSTRACT

A pneumatic bioreactor includes a vessel containing a fluid to be mixed and at least one mixing device driven by gas pressure. A first embodiment includes a floating impeller that rises and falls in the fluid as gas bubbles carry it upward to the surface where the gas is then vented, permitting the impeller to sink in the fluid. The floating impeller may be tethered to a second impeller with a flexible member and pulley. The mixing speed is controlled with electromagnets in the vessel acting upon magnetic material in the impeller or its guides. In another embodiment, floating pistons mix the fluid, pushing it through a mixing plate with one or more apertures. In a third embodiment, the mixing device is a rotating drum with bubble-catching blades and rotating mixing plates with apertures. The top of the vessel for these mixers may include a closed top and sterile filters.

RELATED APPLICATIONS

This application is a continuation application claiming priority under35 U.S.C. § 120 of U.S. patent application Ser. No. 14/444,695 filedJul. 28, 2014, now issued as U.S. Pat. No. 9,453,194, which is acontinuation application claiming priority under 35 U.S.C. § 120 of U.S.patent application Ser. No. 12/884,431 filed Sep. 17, 2010, now issuedas U.S. Pat. No. 8,790,913, which is a continuation-in-part applicationclaiming priority under 35 U.S.C. § 120 of U.S. patent application Ser.No. 11/739,089 filed Apr. 23, 2007, now abandoned.

TECHNICAL FIELD

The invention pertains to apparatus for mixing solutions. Moreparticularly, the invention relates to methods for using pneumaticallyoperated mixers for use in closed, sterile environments.

BACKGROUND OF THE INVENTION

Efforts of biopharmaceutical companies to discover new biological drugshave increased exponentially during the past decade-and-a-half.Bioreactors have been used for cultivation of microbial organisms forproduction of various biological or chemical products in thepharmaceutical, beverage, and biotechnological industry. Most biologicaldrugs are produced by cell culture or microbial fermentation processeswhich require sterile bioreactors and an aseptic culture environment. Anincreasing number of biological drug candidates are in development.Stringent testing, validation, and thorough documentation of process foreach drug candidate are required by FDA to ensure consistency of thedrug quality used for clinical trials to the market. However, shortagesof global biomanufacturing capacity are anticipated in the foreseeablefuture, particularly as production needs will increase as such new drugsare introduced to the market.

A production bioreactor contains culture medium in a sterile environmentthat provides various nutrients required to support growth of thebiological agents of interest. Stainless steel stir tanks have been theonly option for large scale production of biological products insuspension culture. Manufacturing facilities with conventional stainlessbioreactors, however, require large capital investments forconstruction, high maintenance costs, long lead times, andinflexibilities for changes in manufacturing schedules and productioncapacities. Conventional bioreactors use mechanically driven impellorsto mix the liquid medium during cultivation. The bioreactors can bereused for the next batch of biological agents after cleaning andsterilization of the vessel. The procedure of cleaning and sterilizationrequires a significant amount of time and resources, especially tomonitor and to validate each cleaning step prior to reuse for productionof biopharmaceutical products. Due to the high cost of construction,maintenance and operation of the conventional bioreactors, single usebioreactor systems made of disposable plastic material have become anattractive alternative.

While several mixing methods of liquid in disposable bioreactors havebeen proposed in recent years, none of them provides efficient mixingfor large scale (greater than 1000 liters) without expensive operatingmachinery. For this reason, a number of non-invasive and/or disposablemixing systems that do not require an external mechanical operation havebeen developed. Many of these systems work well within certain sizeranges, however, problems sometimes arise as larger mixing systems areattempted.

Single use disposable bioreactor systems have been introduced to marketas an alternative choice for biological product production. Such devicesprovide more flexibility on biological product manufacturing capacityand scheduling, avoid risking major upfront capital investment, andsimplify the regulatory compliance requirements by eliminating thecleaning steps between batches. However, the mixing technology of thecurrent disposable bioreactor system has limitations in terms ofscalability to sizes beyond 200 liters and the expense of large scaleunits. Therefore, a disposable single use bioreactor system which isscalable beyond 1000 liters, simple to operate, and cost effective willbe needed as a substitute for conventional stainless steel bioreactorsfor biopharmaceutical research, development, and manufacturing.

It is an objective of the present invention to provide a pneumaticbioreactor that is capable of efficiently and thoroughly mixingsolutions without contamination. It is a further objective to such areactor that can be scaled to relatively large sizes using the sametechnology. It is a still further objective of the invention to abioreactor that can be produced in a disposable form. It is yet afurther objective of the invention to provide a bioreactor that can beaccurately controlled by internal pneumatic force, as to speed andmixing force applied to the solution without creating a foaming problem.Finally, it is an objective to provide a bioreactor that is simple andinexpensive to produce and to operate while fulfilling all of thedescribed performance criteria.

SUMMARY OF THE INVENTION

A pneumatic bioreactor providing all of the desired features can beconstructed from the following components. A containment vessel isprovided. The vessel has a top, a closed bottom, a surrounding wall andis of sufficient size to contain a fluid to be mixed and a mixingapparatus. The mixing apparatus includes at least one gas supply line.The supply line terminates at an orifice adjacent the bottom of thevessel. At least one buoyancy-driven mixing device is provided. Themixing device moves in the fluid as gas from the supply line isintroduced into and vented from the mixing device. When gas isintroduced into the gas supply line the gas will enter the mixing deviceand cause the device to mix the fluid.

In a variant of the invention, the buoyancy-driven mixing device furtherincludes at least one floating plunger. The plunger has a central,gas-holding chamber and a plurality of mixing elements located about thecentral chamber. The mixing elements are shaped to cause the plunger toagitate the fluid as the plunger rises in the fluid in the containmentvessel. In a variant, the mixing elements are generally in the shape ofa disc.

In yet another variant, the buoyancy-driven mixing device furtherincludes at least one floating impeller, which is also provided as amixing element. The impeller has the central, gas-containing chamber anda plurality of impeller blades arcurately located about the centralchamber. The impeller blades are shaped to cause the impeller to revolveabout a vertical axis as the impeller rises in fluid in the containmentvessel.

The central chamber has a gas-venting valve. The valve permits escape ofgas as the central chamber reaches a surface of the fluid. Aconstraining member is provided. The constraining member limitshorizontal movement of the floating plunger and/or impeller(“plunger/impeller”) as it rises or sinks in the fluid. When gas isintroduced into the gas supply line, the gas will enter the gas-holdingchamber and cause the floating plunger/impeller to rise by buoyancy inthe fluid while agitating the fluid. When the gas-venting valve of thecentral chamber reaches the surface of the fluid, the gas will bereleased and the floating plunger/impeller will sink toward the bottomof the containment vessel where the central chamber will again be filledwith gas, causing the floating plunger/impeller to rise.

In a further variant, a mixing partition is provided. The partition islocated in the containment vessel adjacent the floating plunger/impellerand has at least one aperture to augment a mixing action of the floatingplunger/impeller.

In another variant, means are provided for controlling a rate of assentof the floating plunger/impeller.

In still another variant, the means for controlling the rate of assentof the floating plunger/impeller includes a ferromagnetic substanceattached to either of the floating plunger/impeller, the constrainingmember, or the outside housing, and a controllable electromagnet locatedadjacent the bottom of the containment vessel. The gas flow isinterrupted by an on/off switch which is controlled by interactions oftwo magnetic substances. Therefore, the volume of gas supplied into thegas-holding chamber is determined by the strength of the electromagneticpower since the gas flow stops as the floating plunger/impeller startsto rise when the buoyancy becomes greater than the magnetic holdingforce.

In yet another variant, the central, gas-holding chamber furtherincludes an opening. The opening is located at an upper end of thechamber. A vent cap is provided. The vent cap is sized and shaped toseal the opening when moved upwardly against it by buoyancy from gasfrom the supply line. A support bracket is provided. The support bracketis located within the chamber to support the vent cap when it is loweredafter release of gas from the chamber. When the chamber rises to thesurface of the fluid the vent cap will descend from its weight and theopening will permit the gas to escape, the chamber will then sink in thefluid and the vent cap will again rise due to buoyancy from a smallamount of gas permanently enclosed in the vent cap, thereby sealing theopening.

In a further variant, a second floating plunger/impeller is provided. Asecond constraining member is provided, limiting horizontal movement ofthe second plunger/impeller as it rises in the fluid. At least oneadditional gas supply line is provided. The additional supply lineterminates at an orifice adjacent the bottom of the vessel. At least onepulley is provided. The pulley is attached to the bottom of thecontainment vessel. A flexible member is provided. The flexible memberattaches the chamber of the floating plunger to a chamber of the secondfloating plunger/impeller. The flexible member is of a length permittingthe gas venting valve of the chamber of the floating plunger/impeller toreach the surface of the fluid while the chamber of the second floatingplunger/impeller is spaced from the bottom of the containment vessel.When the floating plunger/impeller is propelled upwardly by buoyancyfrom the gas from the supply line the second floating plunger/impelleris pulled downwardly by the flexible member until the gas is releasedfrom the chamber of the floating plunger/impeller as its gas ventingvalve reaches the surface of the fluid. The chamber will then sink inthe fluid as the second floating plunger/impeller rises by buoyancy fromgas introduced from the second supply line.

In yet a further variant, the containment vessel is formed of resilientmaterial, the material is sterilizable by gamma irradiation methods.

In still another variant, the pneumatic bioreactor further includes acylindrical chamber. The chamber has an inner surface, an outer surface,a first end, a second end and a central axis. At least one mixing plateis provided. The mixing plate is attached to the inner surface of thechamber. First and second flanges are provided. The flanges are mountedto the cylindrical chamber at the first and second ends, respectively.First and second pivot points are provided. The pivot points areattached to the first and second flanges, respectively and to thecontainment vessel, thereby permitting the cylindrical chamber to rotateabout the central axis. A plurality of gas-holding members are provided.The members extend from the first flange to the second flange along theouter surface of the cylindrical chamber and are sized and shaped toentrap gas bubbles from the at least one gas supply line. The gas supplyline terminates adjacent the cylindrical chamber on a first side of thechamber below the gas-holding members. When gas is introduced into thecontainment vessel through the supply line it will rise in the fluid andgas bubbles will be entrapped by the gas-holding members. This willcause the cylindrical chamber to rotate on the pivot points in a firstdirection and the at least one mixing plate to agitate the fluid.

In yet another variant, a rate of rotation of the cylindrical chamber iscontrolled by varying a rate of introduction of gas into the gas supplyline.

In a further variant, a second gas supply line is provided. The secondsupply line terminates adjacent the cylindrical chamber on a second,opposite side of the chamber below the gas holding members. Gas from thesecond supply line causes the cylindrical chamber to rotate on the pivotpoints in a second, opposite direction.

In still a further variant, the at least one mixing plate has at leastone aperture to augment mixing of the fluid in the containment vessel.

In yet a further variant, the containment vessel further includes aclosable top. The top has a vent, permitting the escape of gas from thegas supply line through a sterile filter.

In another variant of the invention, a temperature control jacket isprovided. The jacket surrounds the containment vessel.

In a variant of the invention, an outside housing is provided. Thehousing is ring-shaped and surrounds the floating impeller andconstrains its lateral movement. At least one supporting pole isprovided. The pole extends from the bottom upwardly toward the top. Theoutside housing is slidably attached to the supporting pole. Thefloating impeller is rotatably attached to the outside housing.

In still another variant, the impeller blades are rotatably mounted tothe central chamber and the central chamber is fixedly attached to theoutside housing.

In a further variant, the impeller blades are fixedly mounted to thecentral chamber and rotatably mounted to the outside housing.

In still a further variant, the outside housing further includes ahorizontal interior groove located on an inner surface of the housing.The impeller blades include a projection, sized and shaped to fitslidably within the groove.

In yet another variant, the vent cap further includes an enclosed gascell. The cell causes the cap to float in the fluid and thereby toreseal the opening after the gas has been released when the chamberreached the surface of the fluid.

In a further variant, wherein the pneumatic bioreactor further includesa second floating impeller, a second outside housing surrounding thesecond floating impeller is provided. At least one additional supportingpole is provided. At least one additional gas supply line is provided.The additional supply line terminates at an orifice at the bottom of thevessel. The second outside housing is slidably attached to theadditional supporting pole. The second floating impeller is rotatablyattached to the second outside housing. At least one pulley is provided.The pulley is attached to the bottom of the containment vessel.

An appreciation of the other aims and objectives of the presentinvention and an understanding of it may be achieved by referring to theaccompanying drawings and the detailed description of a preferredembodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a first embodiment of the inventionillustrating floating impellers and their control mechanisms.

FIG. 2 is a top view of the FIG. 1 embodiment illustrating the floatingchamber affixed to the constraining member with the impeller bladesrotating upon the chamber.

FIG. 2A is a top view of the FIG. 1 embodiment illustrating the floatingchamber rotating within the constraining member with the impeller bladesfixed to the chamber.

FIG. 3 is a side elevational view of the FIG. 1 embodiment.

FIG. 4 is a side elevational view of the FIG. 2A embodiment of thefloating impeller.

FIG. 4A is a side elevational view of the FIG. 2 embodiment of thefloating impeller.

FIG. 5 is a perspective view of a second embodiment of the inventionillustrating floating plungers and their control mechanisms.

FIG. 6 is a top view of the FIG. 5 embodiment illustrating the floatingplungers.

FIG. 7 is a perspective view of the gas supply line and magnetic assentcontrol mechanism.

FIG. 8 is a cross-sectional side elevation of the floating chamberillustrating the vent cap in a closed position.

FIG. 9 is a cross-sectional side elevation of the floating chamberillustrating the vent cap in an open position.

FIG. 10 is a perspective view of a third embodiment of the inventionillustrating a rotating drum mixer with gas supply line.

FIG. 11 is an end view of the FIG. 10 embodiment illustrating a singlegas supply line.

FIG. 12 is an end view of the FIG. 10 embodiment illustrating a pair ofgas supply lines.

FIG. 13 is a side elevational view of the FIG. 10 embodimentillustrating a containment vessel.

FIG. 14 is a perspective view of the FIG. 5 embodiment illustrating aclosable top and sterile filters.

FIG. 15 is a perspective view of the FIG. 5 embodiment illustrating atemperature control jacket surrounding the vessel.

FIG. 16 is a perspective view of a pneumatic bioreactor shown through atransparent housing and containment vessel for clarity.

FIG. 17 is a front view of the pneumatic bioreactor of FIG. 16.

FIG. 18 is top view of the pneumatic bioreactor of FIG. 16.

FIG. 19 is a perspective view of the top and mixing apparatus of thepneumatic bioreactor of FIG. 16.

FIG. 20 is a perspective view of one wheel of the pneumatic bioreactorof FIG. 16.

FIG. 21 is a perspective view of the top and mixing apparatus of amodified bioreactor of FIG. 16.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A pneumatic bioreactor 10, as illustrated in FIGS. 1-3, providing all ofthe desired features can be constructed from the following components. Acontainment vessel 15 is provided. The vessel 15 has a top 20, a closedbottom 25, a surrounding wall 30 and is of sufficient size to contain afluid 35 to be mixed and a mixing apparatus 40. The mixing apparatus 40includes at least one gas supply line 45. The supply line 45 terminatesat an orifice 50 adjacent the bottom 25 of the vessel 15. At least onebuoyancy-driven mixing device 55 is provided. The mixing device 55 movesin the fluid 35 as gas 60 from the supply line 45 is introduced into andvented from the mixing device 55. When gas 60 is introduced into the gassupply line 45 the gas 60 will enter the mixing device 55 and cause thedevice to mix the fluid 35.

In a variant of the invention, the buoyancy-driven mixing device 55further includes at least one floating mixer 65. The mixer 65 has acentral, gas-holding chamber 70 and a plurality of mixing elements 75located about the central chamber 70. The mixing elements 75 are shapedto cause the mixer 65 to agitate the fluid 35 as the mixer 65 rises inthe fluid 35 in the containment vessel 15. The central chamber 70, asillustrated in FIGS. 8 and 9, has a gas-venting valve 80. The valve 80permits escape of gas 60 as the central chamber 70 reaches a surface 85of the fluid 35. A constraining member 90 is provided. The constrainingmember 90 limits horizontal movement of the floating mixer 65 as itrises or sinks in the fluid 35. When gas 60 is introduced into the gassupply line 45, the gas 60 will enter the gas holding chamber 70 andcause the floating mixer 65 to rise by buoyancy in the fluid 35 whileagitating the fluid 35. When the gas venting valve 80 of the centralchamber 70 reaches the surface 85 of the fluid 35, the gas 60 will bereleased and the floating mixer 65 will sink toward the bottom 25 of thecontainment vessel 15 where the central chamber 70 will again be filledwith gas 60, causing the floating mixer 65 to rise.

In another variant, means 95, as illustrated in FIG. 7, are provided forcontrolling a rate of assent of the floating mixer 65.

In still another variant, the means 95 for controlling the rate ofassent of the floating mixer 65 includes a ferromagnetic substance 100attached to either of the floating mixer 65 or the constraining member90 and a controllable electromagnet 105 located adjacent the bottom 25of the containment vessel 15.

In yet another variant, as illustrated in FIGS. 8 and 9, the central,gas-holding chamber 70 further includes an opening 110. The opening 110is located at an upper end 115 of the chamber 70. A vent cap 117 isprovided. The vent cap 117 is sized and shaped to seal the opening 110when moved upwardly against it by buoyancy from gas 60 from the supplyline 45. A support bracket 120 is provided. The support bracket 120 islocated within the chamber 70 to support the vent cap 115 when it islowered after release of gas 60 from the chamber 70. When the chamber 70rises to the surface 85 of the fluid 35 the vent cap 115 will descendfrom its weight and the opening 110 will permit the gas 60 to escape,the chamber 70 will then sink in the fluid 35 and the vent cap 115 willagain rise due to buoyancy from a small amount of gas 60 permanentlyenclosed in the vent cap 115, thereby sealing the opening 110.

In a further variant, as illustrated in FIGS. 1-3, a second floatingmixer 125 is provided. A second constraining member 130 is provided,limiting horizontal movement of the second mixer 125 as it rises in thefluid 35. At least one additional gas supply line 135 is provided. Theadditional supply line 135 terminates at an orifice 143 adjacent thebottom 25 of the vessel 15. At least one pulley 140 is provided. Thepulley 140 is attached to the bottom 25 of the containment vessel 15. Aflexible member 145 is provided. The flexible member 145 attaches thechamber 70 of the floating mixer 65 to a chamber 150 of the secondfloating mixer 125. The flexible member 145 is of a length permittingthe gas venting valve 80 of the chamber 70 of the floating mixer 65 toreach the surface 85 of the fluid 35 while the chamber 70 of the secondfloating mixer 125 is spaced from the bottom 25 of the containmentvessel 15. When the floating mixer 65 is propelled upwardly by buoyancyfrom the gas 60 from the supply line 45 the second floating mixer 125 ispulled downwardly by the flexible member 145 until the gas 60 isreleased from the chamber 70 of the floating mixer 65 as its gas ventingvalve 80 reaches the surface 85 of the fluid 35. The chamber 70 willthen sink in the fluid 35 as the second floating mixer 125 rises bybuoyancy from gas 60 introduced from the second supply line 135.

In yet a further variant, the containment vessel 15 is formed ofresilient material 155, the material is sterilizable by gammairradiation methods.

In still a further variant, as illustrated in FIGS. 5 and 6, thebuoyancy-driven mixing device 10 further includes at least one floatingplunger 160. The plunger 160 has a central, gas-holding chamber 70 andat least one disk 165 located about the central chamber 70. The disk 165is shaped to cause the plunger 160 to agitate the fluid 35 as theplunger 160 rises in the fluid 35 in the containment vessel 15. Thecentral chamber 70 has a gas-venting valve 80. The valve 80 permitsescape of gas 60 as the central chamber 70 reaches a surface 85 of thefluid 35. A mixing partition 170 is provided. The partition 170 islocated in the containment vessel 15 adjacent the floating plunger 160and has at least one aperture 175 to augment a mixing action of thefloating plunger 160. A constraining member 180 is provided. Theconstraining member 180 limits horizontal movement of the plunger 160 asit rises or sinks in the fluid 35. When gas 60 is introduced into thegas supply line 45 the gas 60 will enter the gas holding chamber 70 andcause the floating plunger 160 to rise by buoyancy in the fluid 35 whileagitating the fluid 35. When the gas venting valve 80 of the centralchamber 70 reaches the surface 85 of the fluid 35, the gas 60 will bereleased and the floating plunger 160 will sink toward the bottom 25 ofthe containment vessel 15 where the central chamber 70 will again befilled with gas 60, causing the floating plunger 160 to rise.

In another variant of the invention, a second floating plunger 185 isprovided. A second constraining member 190 is provided, limitinghorizontal movement of the second plunger 185 as it rises in the fluid35. At least one additional gas supply line 135 is provided. Theadditional supply line 135 terminates at an orifice 143 adjacent thebottom 25 of the vessel 15. At least one pulley 140 is provided. Thepulley 140 is attached to the bottom 25 of the containment vessel 15. Aflexible member 145 is provided. The flexible member 145 attaches thechamber 70 of the floating plunger 160 to a chamber of the secondfloating plunger 185. The flexible member 145 is of a length permittingthe gas venting valve 80 of the chamber 70 of the floating plunger 160to reach the surface 85 of the fluid 35 while the chamber 70 of thesecond floating plunger 185 is spaced from the bottom 25 of thecontainment vessel 15. The mixing partition 170 is located between thefloating plunger 160 and the second floating plunger 185. When thefloating plunger 160 is propelled upwardly by buoyancy from the gas 60from the supply line 45 the second floating plunger 185 is pulleddownwardly by the flexible member 145 until the gas 60 is released fromthe chamber 70 of the floating plunger 160 as its gas venting valve 80reaches the surface 85 of the fluid 30. The floating plunger 160 willthen sink in the fluid 35 as the second floating plunger 185 rises bybuoyancy from gas 60 introduced from the second supply line 135.

In still another variant, as illustrated in FIGS. 10-13, the pneumaticbioreactor 10 further includes a cylindrical chamber 195. The chamber195 has an inner surface 200, an outer surface 205, a first end 210, asecond end 215 and a central axis 220. At least one mixing plate 225 isprovided. The mixing plate 225 is attached to the inner surface 200 ofthe chamber 195. First 230 and second 235 flanges are provided. Theflanges 230, 235 are mounted to the cylindrical chamber 195 at the first210 and second ends 215, respectively. First 240 and second 245 pivotpoints are provided. The pivot points 240, 245 are attached to the first230 and second 235 flanges, respectively and to the containment vessel15, thereby permitting the cylindrical chamber 195 to rotate about thecentral axis 220. A plurality of gas holding members 250 are provided.The members 250 extend from the first flange 230 to the second flange235 along the outer surface 205 of the cylindrical chamber 195 and aresized and shaped to entrap gas bubbles 255 from the at least one gassupply line 45. The gas supply line 45 terminates adjacent thecylindrical chamber 195 on a first side 260 of the chamber 195 below thegas holding members 250. When gas 60 is introduced into the containmentvessel 15 through the supply line 45 it will rise in the fluid 35 andgas bubbles 255 will be entrapped by the gas holding members 250. Thiswill cause the cylindrical chamber 195 to rotate on the pivot points240, 245 in a first direction 262 and the at least one mixing plate 225to agitate the fluid 35.

In yet another variant, a rate of rotation of the cylindrical chamber195 is controlled by varying a rate of introduction of gas 60 into thegas supply line 45.

In a further variant, as illustrated in FIG. 12, a second gas supplyline 135 is provided. The second supply line 135 terminates adjacent thecylindrical chamber 195 on a second, opposite side 265 of the chamber195 below the gas holding members 250. Gas 60 from the second supplyline 135 causes the cylindrical chamber 195 to rotate on the pivotpoints 240, 245 in a second, opposite direction 270.

In still a further variant, as illustrated in FIGS. 10 and 13, the atleast one mixing plate 225 has at least one aperture 275 to augmentmixing of the fluid 35 in the containment vessel 15.

In yet a further variant, as illustrated in FIG. 14, the containmentvessel 15 further includes a closable top 280. The top has a vent 285,permitting the escape of gas 60 from the gas supply line 45 through asterile filter 290.

In another variant of the invention, as illustrated in FIG. 15, atemperature control jacket 295 is provided. The jacket 295 surrounds thecontainment vessel 15.

In yet another variant, as illustrated in FIGS. 1-3, a pneumaticbioreactor 10 includes a containment vessel 15. The vessel 15 has a top20, a closed bottom 25, a surrounding wall 30 and is of sufficient sizeto contain a fluid 35 to be mixed and a mixing apparatus 40. The mixingapparatus 40 includes at least one gas supply line 45. The supply line45 terminates at an orifice 50 at the bottom 25 of the vessel 15. Atleast one floating impeller 300 is provided. The impeller 300 has acentral, gas-containing chamber 70 and a plurality of impeller blades305 arcurately located about the central chamber 70. The impeller blades305 are shaped to cause the impeller 300 to revolve about a verticalaxis 310 as the impeller 300 rises in fluid 35 in the containment vessel15.

The central chamber 70 has a gas-venting valve 80. The valve 80 permitsescape of gas 60 as the central chamber 70 reaches a surface 85 of thefluid 35. An outside housing 315 is provided. The housing 315 isring-shaped and surrounds the floating impeller 300 and constrains itslateral movement. At least one supporting pole 320 is provided. The pole320 extends from the bottom 25 upwardly toward the top 20. The outsidehousing 315 is slidably attached to the supporting pole 320. Thefloating impeller 300 is rotatably attached to the outside housing 315.When gas 60 is introduced into the gas supply line 45 the gas 60 willenter the gas containing chamber 70 and cause the floating impeller 300to rise in the fluid 35 while rotating and mixing the fluid 35. When thegas venting valve 80 of the central chamber 70 reaches the surface 85 ofthe fluid 35, the gas 60 will be released and the floating impeller 300will sink toward the bottom 25 of the containment vessel 15 where thecentral chamber 70 will again be filled with gas 60, causing thefloating impeller 300 to rise.

In still another variant, as illustrated in FIGS. 2 and 4A, the impellerblades 305 are rotatably mounted to the central chamber 70 and thecentral chamber 70 is fixedly attached to the outside housing 315.

In a further variant, as illustrated in FIGS. 2A and 4, the impellerblades 305 are fixedly mounted to the central chamber 70 and rotatablymounted to the outside housing 315.

In still a further variant, the outside housing 315 further includes ahorizontal interior groove 322 located on an inner surface 325 of thehousing 315. The impeller blades 305 include a projection 330, sized andshaped to fit slidably within the groove 322.

In yet a further variant, as illustrated in FIG. 7, means 95 areprovided for controlling a rate of assent of the floating impeller 300.

In another variant of the invention, the means 95 for controlling a rateof assent of the floating impeller 300 includes a ferromagneticsubstance 100 attached to either the floating impeller 300 or theoutside housing 315 and a controllable electromagnet 105 locatedadjacent the bottom 25 of the containment vessel 15.

In still another variant, as illustrated in FIGS. 8 and 9, the central,gas-containing chamber 70 further includes an opening 110 located at anupper end 115 of the chamber 70. A vent cap 115 is provided. The ventcap 115 is sized and shaped to seal the opening 110 when moved upwardlyagainst it by pressure from gas 60 from the supply line 45. A supportbracket 120 is provided. The support bracket 120 is located within thechamber 70 to support the vent cap 115 when it is lowered after releaseof gas 60 from the chamber 70. When the chamber 70 rises to the surfaceof the fluid 35 the vent cap 115 will descend from its weight and theopening 110 will permit the gas 60 to escape. The floating impeller 300will then sink in the fluid 35 and the vent cap 115 will again rise dueto pressure from gas 60 introduced into the chamber 70 from the gas line45, thereby sealing the opening 110.

In yet another variant, the vent cap 115 further includes an enclosedgas cell 310. The cell 310 causes the cap 115 to float in the fluid 35and thereby to reseal the opening 110 after the gas 60 has been releasedwhen the chamber 70 reached the surface 85 of the fluid 35.

In a further variant, as illustrated in FIGS. 1 and 3, the pneumaticbioreactor 10 further includes a second floating impeller 317. A secondoutside housing 324 surrounding the second floating impeller 317 isprovided. At least one additional supporting pole 326 is provided. Atleast one additional gas supply line 135 is provided. The additionalsupply line 135 terminates at an orifice 143 at the bottom 25 of thevessel 15. The second outside housing 324 is slidably attached to theadditional supporting pole 325. The second floating impeller 317 isrotatably attached to the second outside housing 324. At least onepulley 140 is provided. The pulley 140 is attached to the bottom 25 ofthe containment vessel 15.

A flexible member 145 is provided. The flexible member 145 attaches thechamber 70 of the floating impeller 300 to a chamber 70 of the secondfloating impeller 317. The flexible member 145 is of a length to permitthe gas venting valve 80 of the chamber 70 of the floating impeller 300to reach the surface 85 of the fluid 35 while the chamber 70 of thesecond floating impeller 317 is spaced from the bottom 25 of thecontainment vessel 15. When the floating impeller 300 is propelledupwardly by pressure from the gas 60 from the supply line 45 the secondfloating impeller 315 will be pulled downwardly by the flexible member145 until the gas 60 is released from the chamber 70 of the floatingimpeller 300 as its gas venting valve 80 reaches the surface 85 of thefluid 35, the floating impeller 300 will then sink in the fluid 35 asthe second floating impeller 315 rises under pressure from gas 60introduced from the second supply line 135.

FIGS. 16 through 20 illustrate a bioreactor positioned in a housing,generally designated 410. The housing 410 is structural and preferablymade of stainless steel to include a housing front 412, housing sides414 and a housing back 416. The housing back 416 does not extend fullyto the floor or other support in order that access may be had to theunderside of the bioreactor. The housing 410 includes a housing bottom418 which extends from the housing sides 414 in a semi-cylindrical curveabove the base of the housing 410. One of the front 412 or back 416 mayact as a door to facilitate access to the interior of the housing 410.

The bioreactor includes a containment vessel, generally designated 420,defined by four vessel sides 422, 424, 426, 428, a semi-cylindricalvessel bottom 430, seen in FIG. 17, and a vessel top 432. Two of thevessel sides 424, 428 which are opposed each include a semicircular end.The other two vessel sides 422, 426 join with the semi-cylindricalvessel bottom 430 to form a continuous cavity between the two vesselsides 424, 428. All four vessel sides 422, 424, 426, 428 extend to andare sealed with the vessel top 432 to form a sealed enclosure. Thevessel top 432 extends outwardly of the four vessel sides 422, 424, 426,428 so as to rest on the upper edges of the structural housing front412, sides 414 and back 416. Thus, the containment vessel 420 hangs fromthe top 432 in the housing 410. The vessel, with the exception of thevessel top 432, is of thin wall film which is not structural in nature.Therefore, the housing front 412, sides 414, back 416 and bottom 418structurally support the containment vessel 420 depending from thevessel top 432 when filled with liquid. All joints of the containmentvessel 420 are welded or otherwise sealed to provide the appropriatesealed enclosure which can be sterilized and closed ready for use.

The vessel top 432 includes access ports 434 for receipt or extractionof liquids, gases and powders and grains of solid materials. The accessports 436 in the vessel top 432 provide for receipt of sensors toobserve the process. Two orifices 438, 440 are shown at the vesselbottom 430 slightly offset from the centerline to receive propellant gasfor driving the rotational mixer as will be discussed below. Thesemi-cylindrical vessel bottom 430 defining a semi-cylindrical concavitywithin the containment vessel 420 also includes a temperature controlsheet 442 which may include a heater with heating elements, a coolerwith cooling coils, or both as may be employed to raise or lower thetemperature of the contents of the containment vessel 420 during use.Sealed within the enclosure defining the containment vessel 420, struts444 extend downwardly from the vessel top 432 to define a horizontalmounting axis at or close to the axis of curvature defined by thesemi-cylindrical bottom 430.

A mixing apparatus includes a rotatably mounted rotational mixer,generally designated 448. The rotational mixer 448 is a general assemblyof a number of functional components. The structure of the rotationalmixer 448 includes two parallel wheels 450, 452 which are displaced fromone another. These wheels are tied to an axle 454 by spokes 456.Additional stabilizing bars parallel to the axle 54 may be used torigidify the rotational mixer 448.

Each wheel 450, 452 is defined by two parallel plates 460, 462. Theseplates 460, 462 include buoyancy-driven mixing cavities 464 therebetween. These cavities 464 operate to entrap gas supplied from belowthe wheels 450, 452 through the gas supply at orifices 438, 440. Theorifices 438, 440 are offset from being directly aligned with thehorizontal axis of rotation to insure that the buoyancy-driven cavities464 are adequately filled with gas to power the rotational mixer 448 inrotation. In the embodiment of FIGS. 16 through 20, the buoyancy-drivencavity 464 in each one of the wheels 450, 452 are similarly oriented toreceive gas from the orifices 438, 440 at the same time.

Outer paddles 466 are equiangularly placed to extend axially outwardlyfrom the outer parallel plates 460 where they are attached. These outerpaddles 466 can mix the liquid between the rotational mixer 448 andeither side 424, 428. The outer paddles 466 are formed in thisembodiment with a concavity toward the direction of rotation of therotational mixer 448 and are inclined toward the direction of rotationas well such that they are disposed to induce flow entrained withconstituents of the mix in the vessel inwardly toward the axis for flowthrough each wheel 450, 452 with the rotation of the rotational mixer448. The outer paddles 466 may exhibit an inclined orientation on eachof the outer parallel plates 460 such that any induced axial flowthrough each wheel 450, 452 will flow toward the center of therotational mixer 448 in opposite directions. The number of outer paddles466 may be increased from the four shown, particularly when theconstituents of the mix in the vessel are not easily maintained insuspension. The outer paddles 466 may extend close to the vessel bottom430 to entrain constituents of the mix in the vessel which may otherwiseaccumulate on the bottom. Such extensions beyond the wheels 450, 452preferably do not inhibit rotation of the rotational mixer 448 throughactual or close interaction with the vessel wall.

Inwardly of the two wheels 450, 452, vanes 468 may be employed in someembodiments as can best be seen in FIG. 20. These vanes 468 extendaxially inwardly from the inner parallel plates 462 to span the distancethere between. The vanes 468 can also extend to induce flow radiallyoutwardly from the rotational mixer 448 and beyond the rotational mixer448 so as to impact and mix liquid outwardly of the rotational mixer. Aswith the outer paddles 466, the vanes 468 can be used to entrainconstituents that tend to fall and collect on the vessel bottom 430.These vanes 468 may, in some instances not be preferred because of flowresistance or disruption of circulating flow. Empirical analysis isnecessary in this regard depending on such things as rotational mixerspeed, liquid viscosity, space to the vessel walls and the like. Fourvanes 468 are illustrated on each wheel 450, 452 but the number can, aswith the outer paddles 466, be increased or decreased with theperformance of the mix.

Inner paddles 470 also extend axially inwardly from the inner parallelplates 462. These inner paddles 470 are convex facing toward therotational direction and are inclined to draw flow axially through thewheels 450, 452. The inner paddles 470 can enhance radially outward flowwith rotation of the rotational mixer 448 as well at the location showninside of the wheels 450, 452. There can be any practical number ofinner paddles 470, four being shown. Such paddles 470, if configured toextend past the perimeter of the wheels 450, 452, can urge flow off ofthe bottom as well and direct that flow axially outwardly to eitherside.

Located inwardly of each wheel 450, 452 is an impeller having blades472. The two impellers provide principal axial thrust to the flowthrough the wheels 450, 452. The thrust resulting from these blades 472both flow inwardly toward one another in this embodiment. This isadvantageous in creating toroidal flow about the wheels and balanceforces which would otherwise be imposed on the mountings. The placementof the blades 472 may be at other axial locations such as at either ofthe plates 460, 462. Where the impellers act alone, the blades 472 canbe located anywhere from exterior of to interior to the rotational mixerwith appropriate reconfiguration in keeping with slow speed impellerpractice.

The mixing apparatus defined principally by the rotating rotationalmixer 448 is positioned in the containment vessel 420 such that itextends into the semi-cylindrical concavity defined by the vessel bottom430 and is sized, with the outer paddles 466, vanes 468 and innerpaddles 470, to fill the concavity but for sufficient space between themixing apparatus and the vessel sides 424, 428 and bottom 430 to avoidinhibiting free rotation of the rotational mixer 448. In one embodiment,the full extent of the mixing apparatus 426 is on the order of 10%smaller than the width of the cavity in the containment vessel 420 andabout the same ratio for the diameter of the rotational mixer 448 to thesemi-cylindrical vessel bottom 430. This spacing is not critical so longas the mixing apparatus is close enough and with commensurate speed toeffect mixing throughout the concavity. Obviously, empirical testing isagain of value. The liquid preferably does not extend above the mixingapparatus and the volume above the rotational mixer 448 will naturallybe mixed as well.

In operation, the liquid, nutrients and active elements are introducedinto the containment vessel 420 through the ports 434, 436. The level ofmaterial in the vessel 420 is below the top of the rotational mixer 448to avoid the release of driving gas under the liquid surface which maycause foam. Gas is injected through the orifices 438, 440 to becomeentrapped in the buoyancy-driven cavity 464 in the rotational mixer 448.This action drives the rotational mixer 448 in a direction which is seenas clockwise in FIG. 17.

The blades 472 act to circulate the liquid within the containment vessel420 with toroidal flow in opposite directions through the wheels 450,452, radially outwardly from between the wheels 450, 452 and thenradially inwardly on the outsides of the rotational mixer 448 to againbe drawn into the interior of the rotational mixer 448. Mixing withturbulence is desired and the outer paddles 466, the vanes 468 and theinner paddles 470 contribute to the mixing and to the toroidal flowabout each of the wheels 450, 452. The target speed of rotation is onthe order of up to the low tens of rpm to achieve the similar mixingresults as prior devices at 50 to 300 rpm. The difference may reduceshear damage in more sensitive materials. Oxygen may be introduced in aconventional manner as well as part of the driving gas to be mixed fullythroughout the vessel 420 under the influence of the mixing apparatus.

FIG. 21 illustrates a variation on the embodiment of FIGS. 16 through20. In this embodiment, the buoyancy-driven mixing cavities 464 arereversed in one of the wheels 450, 452 for driving in the oppositedirection. Similarly, the orifices 438, 440 are offset to either side ofthe horizontal axis of rotation. The gas through the orifices 438, 440is independently controlled to allow selection of rotation of therotational mixer in either direction.

Thus, an improved pneumatic bioreactor is disclosed. While embodimentsand applications of this invention have been shown and described, itwould be apparent to those skilled in the art that many moremodifications are possible without departing from the inventive conceptsherein. The invention, therefore, is not to be restricted except in thespirit of the appended claims.

An appreciation of the other aims and objectives of the presentinvention and an understanding of it may be achieved by referring to theaccompanying drawings and the detailed description of a preferredembodiment.

What is claimed is:
 1. A system for performing a cell culture growthprocess, comprising: a containment vessel for holding a cell culturemedium, the containment vessel comprising a generally semi-cylindricalbottom wall defining a semi-cylindrical concavity therewithin, thecontainment vessel having a rotatable mixing wheel mounted within forrotation about a horizontal axis approximately coincident with an axisof curvature of the semi-cylindrical bottom wall, the mixing wheelhaving a rotational diameter that extends into close proximity with thesemi-cylindrical bottom wall; and an impeller defined within an outerrim on the mixing wheel configured to generate axial flow of at least aportion of the contents of the containment vessel upon rotation of themixing wheel, wherein the containment vessel may be enclosed andsterilized so that a cell culture medium and nutrients may be providedwithin the containment vessel to support growth of a cell culture withinthe cell culture medium while rotating the mixing wheel about thehorizontal axis to mix the contents of the containment vessel.
 2. Thesystem of claim 1, further comprising a temperature control sheetarranged in contact with the containment vessel bottom wall forcontrolling the temperature of the interior of the containment vesseland support growth of the cell culture.
 3. The system of claim 1,further including a rigid housing into which the containment vesselfits, the housing defining a semi-cylindrical lower section and a lowerportion of the containment vessel being formed of a material that isnon-structural such that the semi-cylindrical lower section of thehousing structurally supports the semi-cylindrical bottom wall of thecontainment vessel.
 4. The system of claim 3, wherein a vessel topextends outwardly of containment vessel sides and rests on upper edgesof the housing so as to suspend the containment vessel within thehousing.
 5. The system of claim 3, wherein the containment vessel isprimarily formed of a thin wall film that is non-structural.
 6. Thesystem of claim 1, wherein the mixing wheel includes two parallel wheelswhich are axially displaced from one another and fixed to rotate on acommon axle by spokes.
 7. The system of claim 1, further including apaddle that extends axially outward from the mixing wheel to mix liquidbetween the mixing wheel and inner sides of the containment vessel. 8.The system of claim 1, further including an air bubble inlet into thelower portion of the containment vessel to pneumatically rotate themixing wheel.
 9. A system for performing a cell culture growth process,comprising: a first containment vessel for holding a cell culturemedium, the first containment vessel comprising a mixing chambertherewithin and having therein a rotatable mixing wheel mounted forrotation about a horizontal axis, the mixing wheel substantially fillinga lower portion of the mixing chamber, the first containment vesselhaving a vessel top and a lower portion formed of a material that isnon-structural; a rigid housing within which the containment vessel issuspended, the housing having upper edges and a bottom wall on which issupported the non-structural lower portion of the containment vessel,and further wherein the housing has side walls that closely surround andsupport peripheral walls of the containment vessel; and a secondcontainment vessel for holding a cell culture medium, the secondcontainment vessel comprising a mixing chamber therewithin and havingtherein a rotatable mixing wheel mounted for rotation about a horizontalaxis, the mixing wheel substantially filling a lower portion of themixing chamber, the second containment vessel having a vessel top and alower portion formed of a material that is non-structural, wherein thefirst and second containment vessels may be enclosed and sterilized sothat a cell culture medium and nutrients may be provided within eachcontainment vessel to support growth of a cell culture within the cellculture medium while rotating the mixing wheels about the horizontalaxis to mix the contents of the respective containment vessel, and thefirst and second containment vessels may be sequentially suspendedwithin the housing to perform a cell culture growth process and thenremoved therefrom.
 10. The system of claim 9, wherein the containmentvessel comprises a generally semi-cylindrical bottom wall defining asemi-cylindrical lower portion of the mixing chamber, the horizontalaxis being approximately coincident with an axis of curvature of thesemi-cylindrical bottom wall and the mixing wheel has a rotationaldiameter that extends into close proximity with the semi-cylindricalbottom wall.
 11. The system of claim 10, wherein the housing bottom wallis semi-cylindrical in shape and supports the semi-cylindrical bottomwall of the containment vessel.
 12. The system of claim 9, furtherincluding an impeller defined within an outer rim on the mixing wheelconfigured to generate axial flow of at least a portion of the contentsof the containment vessel upon rotation of the mixing wheel.
 13. Thesystem of claim 9, further including a paddle that extends axiallyoutward from the mixing wheel to mix liquid between the mixing wheel andinner sides of the containment vessel.
 14. The system of claim 9,further including an air bubble inlet into the lower portion of thecontainment vessel to pneumatically rotate the mixing wheel.
 15. Asystem for performing a cell culture growth process, comprising: acontainment vessel for holding a cell culture medium, the containmentvessel comprising a mixing chamber therewithin and having therein arotatable mixing wheel mounted for rotation about a horizontal axis, themixing wheel substantially filling a lower portion of the mixing chamberadjacent a bottom wall, the mixing wheel having an outer rim connectedto an axle with spokes and having an impeller located radially withinthe outer rim having paddles that extend outward from the outer rim forurging flow off of the bottom wall upon rotation of the mixing wheel,wherein the containment vessel may be enclosed and sterilized so that acell culture medium and nutrients may be provided within the containmentvessel to support growth of a cell culture within the cell culturemedium while rotating the mixing wheel about the horizontal axis to mixthe contents of the containment vessel.
 16. The system of claim 15,wherein the bottom wall of the containment vessel is generallysemi-cylindrical thus defining semi-cylindrical lower portion of themixing chamber, the horizontal axis being approximately coincident withan axis of curvature of the semi-cylindrical bottom wall and the mixingwheel having a rotational diameter that extends the paddles into closeproximity with the semi-cylindrical bottom wall.
 17. The system of claim15, further comprising a temperature control sheet arranged in contactwith the containment vessel bottom wall for controlling the temperatureof the interior of the containment vessel and support growth of the cellculture.
 18. The system of claim 17, wherein the mixing wheel furtherincludes a paddle that extends axially outward from the parallel platesto mix liquid between the mixing wheel and inner sides of thecontainment vessel.
 19. The system of claim 15, wherein the mixing wheelincludes two parallel plates axially spaced from one another and aplurality of mixing elements evenly arranged therebetween around anouter periphery of the mixing wheel.
 20. The system of claim 15, furtherincluding an air bubble inlet into the lower portion of the containmentvessel to pneumatically rotate the mixing wheel.
 21. A system forperforming a cell culture growth process, comprising: a containmentvessel for holding a cell culture medium, the containment vesselcomprising a generally semi-cylindrical bottom wall defining asemi-cylindrical concavity therewithin, the containment vessel having arotatable mixing wheel mounted within for rotation about a horizontalaxis approximately coincident with an axis of curvature of thesemi-cylindrical bottom wall, the mixing wheel having a rotationaldiameter that extends into close proximity with the semi-cylindricalbottom wall; and a rigid housing into which the containment vessel fits,the housing defining a semi-cylindrical lower section and a lowerportion of the containment vessel being formed of a material that isnon-structural such that the semi-cylindrical lower section of thehousing structurally supports the semi-cylindrical bottom wall of thecontainment vessel, wherein the containment vessel may be enclosed andsterilized so that a cell culture medium and nutrients may be providedwithin the containment vessel to support growth of a cell culture withinthe cell culture medium while rotating the mixing wheel about thehorizontal axis to mix the contents of the containment vessel.
 22. Thesystem of claim 21, further comprising a temperature control sheetarranged in contact with the containment vessel bottom wall forcontrolling the temperature of the interior of the containment vesseland support growth of the cell culture.
 23. The system of claim 21,wherein a vessel top extends outwardly of containment vessel sides andrests on upper edges of the housing so as to suspend the containmentvessel within the housing.
 24. The system of claim 21, wherein thecontainment vessel is primarily formed of a thin wall film that isnon-structural.
 25. The system of claim 21, further including a paddlethat extends axially outward from the mixing wheel to mix liquid betweenthe mixing wheel and inner sides of the containment vessel.
 26. Thesystem of claim 21, further including an air bubble inlet into the lowerportion of the containment vessel to pneumatically rotate the mixingwheel.